Compositions and Methods for Controlling Copy Number for a Broad Range of Plasmids and Uses Thereof

ABSTRACT

The present invention provides compositions and methods for controlling the copy number for a broad range of plasmids and uses thereof. Disclosed is a host cell for conditional control of copy number of a plasmid, which host cell comprises a poly(A) polymerase gene that is operably joined to a conditionally inducible promoter, and a method for cloning and stably maintaining a DNA sequence encoding a heterologous polypeptide in the host cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 10/883,459, filed Jul. 1, 2004, which claimspriority to U.S. Provisional Patent Application Ser. No. 60/483,955,filed Jul. 1, 2003. The entire disclosure of all priority applicationsis specifically incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

FIELD OF THE INVENTION

This invention relates to production and use of host cells for controlof replication of plasmids, including, but not limited to, DNA vectors,in the host cells, and to their use for production of DNA, RNA and/orpolypeptides encoded by DNA contained in these plasmids.

BACKGROUND OF THE INVENTION

Plasmids are normally circular, extrachromosomal DNA molecules thatreplicate autonomously within the cells of host organisms. The cells ofmany unicellular organisms, including some bacteria, contain naturallyoccurring wild-type plasmids that contribute various functions to thehost cells such as antibiotic resistance and fertility.

These wild-type plasmids and derivatives of them are the basic tools ofrecombinant DNA technology, providing vehicles for the transformation ofthe cells of host organisms with foreign DNA sequences which code forproduction, within the transformed cells, of corresponding foreignproducts. As used herein, “foreign products” means DNA, RNA and/orpolypeptides that are foreign to the host cells including but notlimited to DNA sequences encoding foreign genes, RNA moleculestranscribed from inserted additional foreign DNA sequences, andpolypeptides encoded by foreign genes.

Recombinant plasmids, similar to the parent plasmids from which they arederived, are capable of autonomous replication within host cells, and onreplication, reproduce not only the DNA sequences of the parent plasmidbut also the inserted additional DNA sequences, including the foreigngenes. During polypeptide synthesis, transcription and translation ofthe DNA sequences of the recombinant plasmids carried within transformedhost cells give rise to the synthesis of foreign RNA and polypeptides.

One factor which affects the yield of synthesized foreign product is thenumber of copies of the foreign gene which are present within thetransformed cells, i.e. the copy number at which the recombinant plasmidis maintained within the host cells, this being defined normally as thenumber of copies of the plasmid per host genome. Generally speaking, thehigher the copy number of the recombinant plasmid the greater is theyield of foreign product. Both low-copy-number plasmids, usuallymaintained within host cells at about 1-10 copies per genome, and highcopy number plasmids, usually maintained at from 11 up to severalhundred copies per genome, are known. The copy number of a givenwild-type replicon is controlled by DNA sequences surrounding andincluding a DNA sequence that defines the origin of replication (“ori”).Thus, hereinafter we refer to high copy number and low copy numberori's.

High copy number plasmids have been used in recombinant systems with aview to obtaining good yields of foreign products. This can lead toundesirable results, however, since many such high copy number plasmidstend not to be maintained stably within transformed cells and may belost from the cells before they can be grown to sufficient levels topermit bulk production of foreign products. For example, the foreignproduct may be toxic and/or inhibit propagation of the transformedcells, or the high copy number plasmids themselves may be inherentlyunstable or recombine with DNA sequences in other copies of therecombinant plasmid that are present in the same cell.

It is known that the copy numbers of some plasmids can be amplifiedabove normal levels by inhibition of protein synthesis, such as, byaddition of protein synthesis inhibitors such as chloramphenicol to thefermentation medium. However, protein synthesis is required forproduction of most gene products, and therefore the inhibitor must beremoved before synthesis of foreign products can take place. The removalof inhibitor requires complicated manipulations and is not alwayspossible.

Other solutions have also been proposed to overcome the problem ofstable maintenance of high copy number plasmids in host cells. Forexample, in U.S. Pat. Nos. 4,487,835; 4,495,287; and 4,499,189,incorporated herein by reference, Uhlin et al. disclosed the use ofmutant plasmids having a temperature-dependent plasmid copy numberpattern such that the plasmid shows a controlled constant plasmid copynumber when host bacteria carrying the plasmid are cultivated at onetemperature and a much higher or totally uncontrolled copy number whenthe host bacteria carrying the plasmid are grown at a differenttemperature. Thus, cells may be propagated to desired production sizeculture at one temperature at which the plasmid replicates at low copynumber and at which its gene products do not significantly inhibit cellgrowth. The temperature may then be altered, greatly increasing theplasmid copy number and also the corresponding production of geneproducts. However, temperature-dependent copy number may be limited toparticular mutant plasmids, which may or may not contain suitablerestriction enzyme cloning sites for a particular foreign DNA sequence.Also, introduction of copy number temperature dependence may introduce asource of instability into the plasmid, and these mutant plasmids may beunstable or subject to loss when cells carrying them are propagated overa prolonged period of time. Another disadvantage of this approach is thefact that higher temperatures may have a negative impact on proteinstability.

In U.S. Pat. No. 5,015,573, Yarranton et al., incorporated herein byreference, disclosed a new class of vectors to solve the problem ofstable low-copy maintenance of the vector while permitting replicationat high copy number under a different set of conditions to produce ahigh yield of gene product. These vectors had two origins ofreplication. When propagated under a first set of conditions,replication takes place using the first ori and results in a low copynumber and stable inheritance of the vector or recombinant vectorcontaining foreign DNA. Then, when propagated under a second set ofconditions, replication takes place using a second, controllable ori,which is under the control of an inducible promoter. In one embodimentof this invention, the natural promoter which promotes transcription ofthe RNA species (RNAII or a similar species) that provides a primer forinitiation of DNA replication by formation of a complex at or near theorigin of replication is replaced by a controllable promoter. If aheterologous cloned gene is also under the control of a controllablepromoter, both replication and expression of the gene are controllablefrom their respective promoters.

Like the invention disclosed by Yarranton, et al. U.S. Pat. No.6,472,177 of Szybalski et al. and U.S. Pat. No. 5,874,259 of Szybalski,both incorporated herein by reference, disclosed compositions andmethods for controlling copy number of a plasmid, including a BACplasmid, wherein the plasmid contains two origins of replication.According to U.S. Pat. No. 6,472,177, which primarily discussescompositions and methods for dual control of both replication andtranscription, “the conditional origin is provided in addition to aorigin of replication that maintains the vector at a single copy percell,” and “the conditional ori could be any ori that functions in thehost cell and is normally inactive until exposed to thereplication-inducing agent.” Thus, neither Yarranton nor Szybalskidisclose compositions or methods for controlling plasmid copy number bycontrolling replication from a single ori.

U.S. Pat. No. 6,165,749 of Sagawa et al., incorporated herein byreference, also discloses vectors and methods for controlling theexpression of a desired gene by a combination of two control mechanisms,i.e., by control of the copy number of the vector containing the geneand by control of transcription of the gene via an inducible promoterattached to an RNA polymerase gene. Use of these two control mechanismsenabled successful expression of a restriction enzyme that was toxic tothe host cell when expressed without control of copy number of thevector containing the gene. In the case of this invention, the controlof plasmid copy number was obtained by placing the RNAII gene, areplication pre-primer for initiation of replication from the plasmidori, under an inducible promoter. Induction of the gene for RNAIIresulted in an increase in copy number of plasmids containing the ori.

While the compositions and methods disclosed in the art providesolutions for controlling copy number of recombinant plasmids forparticular applications, they suffer from certain disadvantages. All ofthe methods are limited to vectors having particular additional geneticelements, genes or other modifications. For example, the method of Uhlinet al. requires the use of a vector containing a particular mutationthat causes temperature-sensitivity. The methods of Yarranton et al. andof Szybalski et al. require the use of vectors with two origins ofreplication, which increases the size of the vector and in most caseswill limit the number and kind of restriction enzyme sites available forcloning of foreign genes. The method of Sagawa is limited to vectorsthat contain particular inducible RNAII-encoding DNA sequences forhigh-copy replication of the vector and, in most cases, an inducible RNApolymerase gene for transcription of a foreign gene that is cloned inthe vector.

What is needed in the art are host cells and methods that enablecopy-number control of replication of a broad range of widely-availableplasmids from ori's that are capable of low-copy replication under oneset of conditions and of high-copy replication under another set ofconditions.

What is needed are host cells and methods that do not requiremodification or genetic engineering of the plasmid in order to controlthe copy number of the plasmid or of recombinant clones made using theplasmid.

In one embodiment, what is needed are host cells and methods for easilymaintaining commonly-used plasmids and plasmid clones at low copy numberfor stable maintenance of clones and minimal loss of cloned DNAsequences that would be toxic or detrimental to the host cell at highcopy number, and yet, which permit the plasmid and plasmid clones to beinduced to high copy number in a tightly-controlled manner by means ofsimple reagents or conditions in order to obtain larger amounts andtherefore, also, a higher purity of foreign products for the desiredapplication.

In an alternate embodiment, what is needed are host cells and methodsfor inducible control of replication and copy number of a broad range ofplasmids that have ori's with antisense-mediated replication controlmechanisms, such as plasmids having ori's of the types contained inColE1- and R1-type plasmids.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is a host cell for controllablychanging the level of replication and copy number of a plasmid that isintroduced into said host cell, wherein said host cell comprises apoly(A) polymerase gene that is operably joined to a conditionallyinducible promoter, and wherein induction of said poly(A) polymerasegene results in a change in the copy number of said plasmid in said hostcell by catalyzing polyadenylation of an antisense RNA molecule thataffects initiation of replication from an origin of replication in saidplasmid.

A primary object of the present invention is to provide host cells andmethods for making and using host cells that enable control of copynumber for a broad range of plasmids, including, but not limited tocommonly used vectors, having an origin of replication that is regulatedby an antisense RNA molecule, wherein polyadenylation of said antisenseRNA molecule affects copy number of the plasmid in said host cell.Another primary object of the present invention is to provide host cellsand methods for making and using said host cells that are capable oflow-copy replication of plasmids having ori's of the present invention,such as, but not limited to, plasmids with ColE1-type ori's, under oneset of conditions and of high-copy replication of these same plasmidsfrom said ori under another set of conditions.

Another object of the present invention is to improve cloning bypermitting control of clone copy number at will. Another object of theinvention is to improve sequencing, particularly high throughputsequencing, by permitting control of clone copy number at-will, mostparticularly by permitting control of clone copy number for clones inBAC, fosmid, and plasmid vectors.

Another primary object of the invention is to provide improved hostcells and methods that permit successful cloning and stable maintenanceat low plasmid copy number per cell of DNA comprising repetitivesequences, or AT-rich or GC-rich sequences, or sequences that are toxicor detrimental for the host cell, including without limitation,sequences that comprise one or more genes that encodes one or morepeptides or proteins which is toxic or detrimental for the host cellwhen expressed. In short, another primary object of the invention is toprovide host systems that permit successful cloning and stablemaintenance of difficult-to-clone sequences at approximately one copyper cell, but which can be easily and rapidly induced to higher copynumber on demand.

It is an object of the present invention to produce heterologouspolypeptides in a host cell, even where the polypeptide is toxic or hasother adverse effects on the host cell that would prevent cloning and/orstable maintenance of inserted polynucleotide prior to theoverproduction of the polypeptide in conventional cell-based proteinexpression systems.

It is a further advantage of the present invention that the host cellsare able to produce large quantities of a heterologous polypeptide, eventoxic polypeptides, because the cells both amplify the plasmid andtranscribe the DNA quickly after induction. Before induction, a leakytranscriptional promoter can have little effect on the cells, becausethe plasmid copy number is so low.

Notations and Nomenclature

The terms used herein have the following meaning with respect to thepresent invention:

As used herein, a “plasmid” is a DNA molecule that can replicateautonomously following its introduction into a host cell, including butnot limited to, a DNA molecule in which other DNA, including, but notlimited to, “foreign” or “heterologous” DNA can be operably joined. Byway of example, but not of limitation, a plasmid of the invention can bea vector, BAC, fosmid, a P1 vector, episome, or any other suitable DNAmolecule.

The words “foreign” or “heterologous” refer to the fact that the DNAwhich is operably joined to the plasmid is not normally present in thehost cell in which it is replicated. The most common method by which theDNA is “operably joined” to a plasmid is by covalent joining ofcompatible ends by means of an enzyme referred to as a ligase, such as,but not limited to, T4 DNA ligase, by a process referred to as“ligation.” However, the invention also includes any other method forjoining or “ligating” foreign DNA into a plasmid including, but notlimited to, chemical joining or joining by methods known in the art thatuse a topoisomerase. The process of ligating a DNA molecule in a plasmidand then replicating this molecule in a host is referred to as “cloning”or “molecular cloning”.

As used herein, an “inducing agent” is a substance that activates thepromoter, either by positively regulating the transcription from thepromoter, or by binding to a repressor that would otherwise inhibittranscription from the promoter. In either case, the inducer activatestranscription from an inducible promoter.

In this application, “operably joined” means that the promoter issituated upstream of the polynucleotide coding sequence such thatproductive transcription of the polynucleotide is initiated at thepromoter. The term “polypeptide” broadly encompasses all proteinaceousmolecules including, without limitation, oligopeptides, peptides andproteins, as those terms are understood in the art.

BRIEF DESCRIPTION OF THE FIGURE

The following FIGURE forms part of the present specification and isincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thisFIGURE in combination with the detailed description of specificembodiments presented herein.

FIG. 1 shows plasmid preparations obtained from control clones that didnot comprise a pncB gene operably joined to a conditionally induciblepromoter (“control”), plasmid preparations from clones that comprised apcnB gene operably joined to a conditionally inducible promoter, whichclones had been grown under conditions that induced an increase ofplasmid copy number through the addition of an inducing agent(“induced”), and plasmid preparations from clones that comprised a pcnBgene operably joined to a conditionally inducible promoter, which cloneshad been grown under conditions that did not induce an increase ofplasmid copy number, because no inducing agent was added (“uninduced”).

The lanes marked with an “M” show a supercoiled molecular weight marker,the arrowheads indicate bands of DNA of 2 kb, 4 kb, and 8 kb. Lane 1shows the plasmid preparation of TransforMax™ EC100™-T1®cells containingpUC19 plasmids (control); lane 2 shows the plasmid preparation ofpcnB-<araBpcnB> Clone#4 cells containing pUC19 plasmids grown withoutthe addition of an inducing agent (uninduced); lane 3 shows the plasmidpreparation of pcnB-<araBpcnB> Clone#4 cells containing pUC19 plasmidsgrown with the addition of arabinose as an inducing agent (induced);lane 4 shows the plasmid preparation of TransforMax™ EC100™-T1®cellscontaining pET11a/rnhA plasmids (control); lane 5 shows the plasmidpreparation of pcnB-<araBpcnB> Clone#4 cells containing pET11a/rnhplasmids grown without the addition of an inducing agent (uninduced);lane 6 shows the plasmid preparation of pcnB-<araBpcnB> Clone#4 cellscontaining pET11a/rnh plasmids grown with the addition of arabinose asan inducing agent (induced); lane 7 shows the plasmid preparation ofTransforMax™ EC100™-T1®cells containing TOPO® TA plasmids (control);lane 8 shows the plasmid preparation of pcnB-<araBpcnB> Clone#4 cellscontaining TOPO® TA plasmids grown without the addition of an inducingagent (uninduced); lane 9 shows the plasmid preparation ofpcnB-<araBpcnB> Clone#4 cells containing TOPO® TA plasmids grown withthe addition of arabinose as an inducing agent (induced); lane 10 showsthe plasmid preparation of TransforMax™ EC100™-T1®cells containingpMOD<pCC-BAC> plasmids (control); lane 11 shows the plasmid preparationof pcnB-<araBpcnB> Clone#4 cells containing pMOD<pCC-BAC> plasmids grownwithout the addition of an inducing agent (uninduced); lane 12 shows theplasmid preparation of pcnB-<araBpcnB> Clone#4 cells containingpMOD<pCC-BAC> plasmids grown with the addition of arabinose as aninducing agent (induced); lane 13 shows the plasmid preparation ofTransforMax™ EC100™-T1®cells containing pHC79 plasmids, which are alsoreferred to as “fosmids” (control); lane 14 shows the plasmidpreparation of pcnB-<araBpcnB> Clone#4 cells containing pHC79 fosmidsgrown without the addition of an inducing agent (uninduced); and lane 15shows the plasmid preparation of pcnB-<araBpcnB> Clone#4 cellscontaining pHC79 fosmids grown with the addition of arabinose as aninducing agent (induced).

DETAILED DESCRIPTION OF THE INVENTION

Two types of mechanisms basically control the replication of plasmidDNA. One utilizes a series of repeated sequences, designated iterons,which are capable of interacting with a protein that initiates DNAreplication. In the other type of mechanism, antisense RNA moleculeshybridize with the RNA molecule that is responsible for the initiationof plasmid replication. The replication of plasmids and mechanisms ofcontrol of replication of plasmids are reviewed by L. Actis et al.(Frontiers in Bioscience, 3: d43-62, 1998), by S. Brantl (Plasmid, 48:165-173, 2002), by G. del Solar and M. Espinosa (Molecular Microbiol.,37: 492-500, 2000), by G. del Solar et al. (Microbiology and MolecularBiology Reviews, 62: 434-464, 1998), and earlier, by Y. Eguchi et al.(Annu. Rev. Biochem., 60: 631-652, 1991) and by K.J. Marians (Annu. Rev.Biochem., 61: 673-719, 1992), all of which are incorporated herein byreference.

The replication of ColE1 and related plasmids is well characterized.ColE1-type plasmids, of which ColE1 plasmid itself is an example, haveplasmid replication systems having a number of features in common. Thesefeatures include a DNA sequence defining an origin of replication andupstream thereof a DNA sequence coding for transcription, in opposingdirections, of two RNA species, RNAII and RNAI. The RNAII speciesprovides an RNA primer, sometimes called a “pre-primer”, that anneals ator near the origin from which DNA synthesis is initiated (forming aninitiation complex), and is then processed by RNase H to generate the3′-end of the replication primer. DNA polymerase I and other hostproteins then replicate the plasmid DNA by extending the RNase Hgenerated end of the RNAII primer. ColE1 plasmids and plasmids having asimilar mechanism for initiation of replication are defined as“ColE1-type” plasmids herein.

The RNAI species, which is complementary to a portion of the RNAIIspecies (i.e., is an antisense molecule to RNAII) interferes with theformation of this initiation complex. Transcription of the two RNAspecies are controlled by separate promoter sequences associated withthe DNA sequences which code for their transcription. Plasmidscontaining a ColE1-type origin of replication are widely used forcloning, for in vitro transcription, and for gene expression purposes inEscherichia coli and in some other prokaryotes.

A second type of mechanism in which antisense RNA molecules hybridizewith mRNA molecules involved in the initiation of plasmid replication isfound in the R1 plasmid. Replication of R1 is initiated by a protein,known as RepA protein, and the RepA mRNA, called CopT (for “Coptarget”), is regulated by an antisense RNA called CopA. CopA controlsinitiation of R1 replication by interacting with CopT, causingposttranscriptional inhibition of RepA synthesis. R1 and plasmids havinga similar mechanism for initiation of replication are defined as“R1-type” plasmids herein. The R1-type of plasmid replication wascharacterized by P. Blomberg et al. (Mol. Microbiol., 12: 49-60, 1994),P. Blomberg et al. (Embo J., 11: 2675-2683, 1992), P. Blomberg et al.(Embo J., 9: 2331-2340, 1990), C. Malmgren et al. (J. Biol. Chem., 272:12508-12512, 1997), K. Nordstrom et al. (Gene, 72: 237-240, 1988), P.Stougaard et al. (Proc. Natl. Acad. Sci. USA, 78: 6008-6012, 1981), E.Wagner et al. (Embo J., 11: 1195-1203, 1992), and C. Malmgren et al.(RNA, 2: 1022-1032, 1996), all of which are incorporated herein byreference.

In addition to the RNAII primer, the RNAI antisense molecule, the CopTmRNA, and the CopA antisense RNA, other molecules are also known thataffect copy number of plasmids with antisense RNA regulated origins ofreplication. By way of example, but not of limitation, a protein encodedby the pcnB gene is known to have an effect on copy number of ColE1-typeplasmids and R1-type plasmids in E. coli (L. Actis et al., Frontiers inBioscience, 3: d43-62, incorporated herein by reference, and F. Soderbomand E. Wagner, Microbiology 144: 1907-1917, 1998, incorporated herein byreference).

The pcnB gene was first identified by J. Lopilato et al. (Mol. Gen.Genet., 205: 285-290, 1986) based on an E. coli chromosomal mutationthat substantially reduced the copy number of ColE1-type plasmids, whichled them to designate the locus causing this as pcnB for “plasmid copynumber”. The E. coli gene was subsequently cloned and sequenced (J.March et al., Mol. Microbiol., 3: 903-910, 1989; and J. Liu and J.Parkinson, J. Bacteriology, 171: 1254-1261, 1989) and later, evidencewas provided that the pcnB locus encodes a poly(A) polymerase protein(G. Cao and N. Sarkar, Proc. Natl. Acad. Sci. USA, 89: 10380-10384,1992). Without being bound by theory, it is believed that that the rapiddegradation of RNAI is associated with polyadenylation of the RNA andthat this process is mediated by poly(A) polymerase (L. He et al., Mol.Microbiol., 9: 1131-1142, 1993; and F. Xu et al., Proc. Natl. Acad. Sci.USA, 90: 6756-6760, 1993). The absence of poly(A) polymeraseintermediates causes the intracellular accumulation of RNAI decayproducts that have antisense activity and can inhibit initiation ofplasmid replication, resulting in a reduced copy number of ColE1-typeplasmids (L. He et al., Mol. Microbiol., 9: 1131-1142, 1993; and F. Xuet al., Proc. Natl. Acad. Sci. USA, 90: 6756-6760, 1993). In addition tomediating the degradation of RNAI, polyadenylation is also believed todecrease the interaction of RNAI with its RNAII target thus affectingthe copy number of ColE1-type plasmids (F. Xu et al., Plasmid, 48:49-58, 2002). M. Masters et al. (J. Bacteriol., 175: 4405-4413, 1993)found that strains deleted for pcnB grow normally demonstrating that thepcnB gene is dispensable in E. coli. All publications cited within thisparagraph are incorporated herein by reference.

The role of the pcnB gene was further studied by U. Binnie et al.(Microbiol., 145: 3089-3100, 1999); F. Xu and S. Cohen (Nature, 374:180-183, 1995); C. Ingle and S. Kushner (Proc. Natl. Acad. Sci. USA, 93:12926-12931, 1996); J. Jasiecki and G. Wegrzyn (EMBO Rep., 4: 172-177,2003); N. Binns and M. Masters (Mol. Microbiol., 44: 1287-1298, 2002);S. Yehudai-Resheff and G. Schuster (Nucleic Acids Res., 28: 1139-1144,2000); L. Raynal and A. Carpousis (Mol. Microbiol., 32: 765-775, 1999);E. Hajnsdorf and P. Regnier (J. Mol. Biol., 286: 1033-1043, 1999); Z. Liet al. (Proc. Natl. Acad. Sci. USA, 95: 12158-12162, 1998); F. Soderbomet al. (Mol. Microbiol., 26: 493-504, 1997); N. Sarkar (Microbiology,142: 3125-3133, 1996); L. Raynal et al. (Biochimie, 78: 390-398, 1996);G. Cao et al. (Proc. Natl. Acad. Sci. USA, 93: 11580-11585, 1996); andM. Masters et al. (Mol. Gen. Genet., 220: 341-344, 1990).

The host cell strain of the present invention is preferably bacterial,preferably an E. coli strain, but the host cell can be any cell in whicha plasmid replicates using an ori from which the level of replication iscontrollable by induction of a poly(A) polymerase gene that is operablyjoined to a conditionally inducible promoter. The host cell can be aplant, yeast or other fungal cell, or even an animal cell, including amammalian cell, as long as the ori is selected so as to function in theselected host. One could also employ shuttle vectors incorporating asuitable ori that functions in the host cell that is used according tothe present invention. C. Hamilton (Gene, 200: 107-116, 1997),incorporated herein by reference, describes a binary-BAC shuttle vectorfor use also in plant cells, but without the presently disclosed plasmidcopy number control feature. A host cell can also be a cell that isgenetically engineered to support replication of a plasmid the copynumber of which is mediated by a poly(A) polymerase that polyadenylatesan antisense RNA that controls replication.

A plasmid used with a host cell of the present invention can be anyplasmid that has an ori wherein initiation of replication from said oriis controlled, at least in part, by an antisense RNA and whereinpolyadenylation of said antisense RNA results in a change in the levelof replication and of the copy number of said plasmid.

In some embodiments, the ori can be an ori wherein replication isinitiated from an RNA pre-primer with which the antisense RNA interacts.By way of example, but not of limitation, plasmids having this type ofori comprise ColE1-type plasmids, including, but not limited to thefollowing, for which the corresponding references are incorporatedherein: ColE1 and pMB1 (F. Bolivar, Life Sci., 25: 807-817, 1979; A.Bhagwat and S. Person, Mol. Gen. Genet., 182: 505-507, 1981), p15A,pJHCMW1 (K. Dery et al., Plasmid, 38: 95-105, 1997; this is a Klebsiellapneumoniae plasmid), pSW100 (J. Fu et al., Plasmid, 34: 75-84, 1995;this is an Erwinia stewartii plasmid), pEC3 (N. Nomura and Y. Murooka,J. Ferment. Bioeng., 78: 250-254, 1994; this plasmid is from Erwiniacarotovora subsp. carotovora), pBR322 and other plasmids of the pBRseries (P. Balbas et al., Biotechnol., 10: 5-41, 1988; L. Covarrubias etal., Gene, 13: 25-35, 1981), of the pUC series of plasmids (J. Viera andJ. Messing, Gene, 19: 259-268, 1982), the pET series (F. Studier, etal., Methods Enzymol., 185: 60-89, 1990), the pBluescript™ series, thepBAD series, a plurality of vectors of the Gateway™ series, a pluralityof vectors of the TOPO® series, pAT153, NTP1, CIoDF13, RSF1030, P15A,and many other vectors that have an ori that is regulated by anantisense RNA molecule, wherein polyadenylation of the antisense RNAmolecule has an effect on the copy number of the plasmid.

The invention is not limited to host cells that are only for induciblemodification of copy number of plasmids with ColE1-type origins ofreplication. Still other plasmids that can be used with host cells ofthe present invention can comprise an ori wherein replication isinitiated by means of a protein that is encoded by an mRNA with whichthe antisense RNA interacts. Thus the invention also comprises hostcells and methods to make and use host cells for plasmids having otherorigins of replication wherein replication from the ori is regulated byan antisense RNA molecule and wherein polyadenylation of an antisenseRNA molecule results in a change in the copy number of the plasmid. Byway of example, but not of limitation, it is also known in the art thatR1-type plasmids regulate initiation of replication via an antisense RNAmolecule (E. Wagner and R. Simons, Annu. Rev. Microbiol., 48: 713-742,1994, incorporated herein by reference).

By way of example, but not of limitation, R1-type plasmids comprise thefollowing plasmids: IncFII plasmids, which include R1, R100, and R6,R6-5, RP4, other related genetic elements belonging to the IncFIIa,IncFIFc, IncFIII, and IncFVII groups, as well as pUB100 and pC194 whichexemplify Staphylococcal plasmids.

The invention comprises any host strain that permits inducible controlof plasmid copy number for any plasmid wherein replication of theplasmid is controlled in some manner by an antisense RNA molecule andwherein inducible control of a poly(A) polymerase in said host cellaffects the copy number of said plasmid in said host cell. Thus, hostcells and methods for controlling copy number of R1-type plasmids arewithin the scope of the present invention.

In preferred embodiments of the invention, if the cell that is used tomake a host cell of the invention has a constitutively-expressed poly(A)polymerase gene that catalyzes polyadenylation of an antisense RNAmolecule that is involved in control of replication from the ori, thisconstitutively-expressed gene is either removed by homologousrecombination or other methods known in the art or is inactivated usinga method such as, but not limited to, transposon insertion, or otherrandom or site-specific mutagenesis techniques that are well known inthe art, and is replaced by a poly(A) polymerase gene that is operablyjoined to an inducible promoter. Preferably, the level of endogenousexpression in the host cell of the poly(A) polymerase thatpolyadenylates the antisense RNA is zero or undetectable.

However, in some embodiments, if the level of an endogenous poly(A)polymerase gene is very low, it may not be essential according to thepresent invention to remove or inactivate an endogenous poly(A)polymerase gene of the host cell. The level of endogenous poly(A)polymerase activity that is acceptable in a particular host cell of theinvention can vary with the effect of poly(A) polymerase activity oncopy number of a particular plasmid, which in turn can vary based onother factors, such as the particular ori used, and the relative levelof the antisense RNA that is involved with regulation of replication. Itwill also be understood by those with knowledge in the art that mutantforms of the poly(A) polymerase can have different activities inpolyadenylation of the antisense RNA and that the acceptable endogenouslevel of expression of the poly(A) polymerase protein in a host cell ofthe invention can vary accordingly.

In an important embodiment of the invention, the controllablereplication systems may be prepared by replacement of the naturalpromoter that promotes transcription of a poly(A) polymerase gene by acontrollable promoter. Alternatively, the natural promoter may be usedand transcription of the poly(A) polymerase made controllable byincorporating a regulating function, such as, but not limited to, anoperator sequence, e.g. the lac operator or O_(L) or O_(R) operators ofphage lambda, into the transcription system.

The poly(A) polymerase gene that is operably joined to a conditionallyinducible promoter in a host cell of the invention can be any gene thatencodes a protein that polyadenylates an antisense RNA that affectsplasmid copy number. A preferred poly(A) polymerase gene of theinvention for E. coli host cells is a pcnB gene that encodes a poly(A)polymerase I protein. Similar genes that encode poly(A) polymerase genesare also known for other cells, some of which a skilled artisan caneasily find by conducting a sequence similarity search of U.S. orinternational sequence databases, such as, but not limited to, a BLAST®(Basic Local Alignment Search Tool) search.

The invention comprises any and all host cells that use any poly(A)polymerase that polyadenylates an antisense RNA that changes the levelof replication and copy number of a plasmid that is introduced into thathost cell.

The invention also comprises mutant forms of poly(A) polymerase genesand their corresponding proteins that are active for the purposes of theinvention, and such mutant genes, and their encoded proteins can bedesirable in some embodiments in order to modify (either increase ordecrease) copy number of particular plasmids or plasmid clones for aparticular purpose. In one aspect of these embodiments said mutant formsof poly(A) polymerase genes have a higher rate of translation than thewildtype poly(A) polymerase gene. In some embodiments of the invention aplurality of poly(A) polymerase genes is inserted into the host cellchromosome which increases the level of expression of poly(A)polymerase.

In most embodiments of host cells of the invention, which are preferredembodiments, the poly(A) polymerase gene that is operably joined to aconditionally inducible promoter is inserted in the chromosome of the ahost cell. However, the invention also envisions embodiments in whichthe poly(A) polymerase gene that is operably joined to a conditionallyinducible promoter is in another extrachromosomal genetic element, suchas in a mitochondrial DNA or in another plasmid that is compatible inthe host cell with the plasmid the copy number of which is changed bysaid conditionally expressed poly(A) polymerase. Preferably, if theconditionally expressible poly(A) polymerase gene is in a plasmid orother extrachromosomal element, the plasmid containing the gene is atlow copy number in the host cell, since it has been shown that inductionof expression of a pcnB-encoded poly(A) polymerase from a multicopyplasmid in an E. coli cell can be toxic to the cell (B. Mohanty and S.Kushner, Mol. Microbiol., 34: 1094-1098, 1999, incorporated herein byreference).

Inducible promoters are known to the art and a detailed summary of thestate of the art is not provided herein. A suitable inducible promoterfunctions in the selected host cell and responds to an inducing agentwith sufficient strength to promote a high level of transcription of adownstream polynucleotide operably joined to the inducibletranscriptional promoter in the plasmid.

Before induction the promoter should normally be inactive, resulting ininsignificant or undetectable levels of product as measured byconventional detection methods in the non-induced state. It is alsopreferred that the promoter require only a single agent for induction.Although the inducible promoter could be any promoter having theseattributes, preferred inducible promoters are the araC-P_(araBAD)(activator gene)-promoter (araC-P_(araBAD); GenBank Accession No. X81838 nt 1-1277) and the TetR/P_(LtetO) repressor promoter (P_(LtetO);GenBank Accession No. U 66312). These promoters are preferable becausethey are tightly regulated when non-induced, and very strong wheninduced. These two promoters can be activated by treating the host cellswith 0.01% L-arabinose (LA) and 100 ng/ml anhydrotetracycline (aTc), (R.Lutz and H. Bujard, Nucleic Acids Res., 25: 1203-1210, 1997,incorporated herein by reference), respectively. Concentrations of LAand aTc shown above are believed optimal but are not essential.AraC/P_(araBAD) also responds to an anti-inducer, D-fucose. Thus, theactivity of AraC/P_(araBAD) can be regulated by adjusting theLA/D-fucose ratio. Other suitable promoters include, but are not limitedto, P_(tac), and a phage promoter under inducible control such as butnot limited to a T3 promoter, a T5 promoter, a T7 promoter, and an SP6promoter.

The origin of replication, the RNA coding sequences and associatedpromoters together, and other genes that encode proteins that affect thelevel, stability, or interaction of antisense RNA molecules with areplication primer or mRNA provide an internally self-regulated systemwhich controls the replication incompatibility and the copy number ofplasmids having a replication system that is controlled by antisenseRNA. The present invention comprises host cells and methods for makingand using these host cells for controlling copy number of plasmidshaving a ColE1-type origin of replication or an R1-type origin ofreplication.

The present invention is not intended to comprise compositions ormethods for controlling copy number plasmids in which control ofreplication from the respective ori does not involve an antisense RNAmolecule. By way of example, compositions or methods for controllingcopy number of plasmids using the oriV origin of replication areexcluded from the scope of the present invention.

Another embodiment of the invention is a host cell that comprises apoly(A) polymerase gene operably joined to a conditionally induciblepromoter, wherein said host cell also comprises another gene, such as,but not limited to a gene encoding an RNA polymerase, that is operablyjoined to a conditionally inducible promoter. In a first aspect of thisembodiment, the conditionally inducible promoter that is operably joinedto another gene, such as, but not limited to, a gene encoding an RNApolymerase, is different from the conditionally inducible promoter towhich the poly(A) polymerase is operationally joined. In a second aspectof this embodiment, the conditionally inducible promoter that isoperably joined to another gene, such as but not limited to, a geneencoding an RNA polymerase, is the same as the conditionally induciblepromoter to which the poly(A) polymerase is operationally joined.

The inducible promoters used for the present invention can be activatedby suitable signals in a host cell of the invention. The inducing agentscan be positive regulators or can interact with negative regulators toincrease amplification and transcription as desired. A positiveregulator (inducer) acts by providing a signal that increases anactivity while a negative regulator (repressor) prevents an activityuntil an agent (also historically designated as inducer) prevents thenegative regulation.

The agents can be organic or inorganic chemical agents or can bepolypeptides encoded by polynucleotide sequences in the host cell genomeor on an extrachromosomal plasmid present in the host cell.Alternatively, the agents can be administered manually to the host cellsby, e.g., providing the agent in the growth medium.

The skilled artisan will appreciate that it is within the level of skillin the art to provide as simple or as complex a regulatory scheme asdesired for ensuring that the appropriate inducing agent is availablefor inducing activity of the poly(A) polymerase or other gene at theappropriate time. The precise nature of that scheme is not critical tothe invention. Rather, for purposes of this invention, it is understoodthat the ultimate agents for amplifying the plasmid and for inducingtranscription can be provided as needed.

Plasmids can be inserted into the host cells of the invention usingstandard nucleic acid transfer methods, such as, but not limited to,electroporation, calcium-mediated transformation or cos-mediated phagelambda packaging and transfection.

By means of processes of the invention, transformed host cells arepropagated to give the cultures required for economic production of DNA,RNA, polypeptides or other products under conditions where the plasmidreplicates at low copy number and the instability problems associatedwith high copy number plasmids are avoided, followed by induction ofreplication wherein the plasmid replicates at high copy number withconcomitant high yield of polynucleotide, polypeptide, protein or otherproducts.

In addition to poly(A) polymerase genes that polyadenylate antisense RNAmolecules that bind to an RNA pre-primer or to an mRNA that encodes aninitiator protein, the invention also envisions that other genes thatare involved in the maturation of an initiator protein or a primerrequired for initiation of replication can be used to make host cellsand methods for conditional control of plasmid copy number. By way ofexample, but not of limitation, a small nonessential polypeptide calledthe Rop or Rom protein appears to increase the efficiency of interactionand stability of interaction between the RNAI antisense molecule and theRNAII replication primer.

Also way of example, but not of limitation, host cells may be made whichhave conditional expression of genes that mediate the maturation of theRNAII primer of ColE1-type plasmids, such as, but not limited to, a genethat encodes a Rop or Rom Protein, a gene that encodes RNase E₁ a genethat encodes RNase H, or a gene that encodes RNAI.

Conditional expression of other genes that accelerate or restrain theformation of an initiator protein or primer may be used in addition toor in place of conditional expression of a poly(A) polymerase gene inorder to make host cells and methods for modulation or fine tuning ofplasmid copy numbers for particular applications. If host cells are madethat permit conditional expression of more than one gene, the expressionof those genes can be from the same inducible promoter or from differentpromoters that are induced independently by different inducers.Applications for using the host cell of the invention for modulation orfine tuning of plasmid copy numbers include but are not limited tofermentation processes involving the use of genetically engineeredbacteria for the production of plasmid DNA, RNA, and recombinantpolypeptides of interest.

It can further be envisioned that the present invention can be used incombination with a method for operably joining an RNAII gene or an RNAIgene to a conditionally inducible promoter for further fine-tuning ofplasmid number control wherein said RNAII gene or said RNAI gene andsaid conditionally inducible promoter can be located on the hostchromosome or another extrachromosomal genetic element, such as inanother plasmid that is compatible in the host cell with the plasmid thecopy number of which is changed by a conditionally expressed poly(A)polymerase.

EXAMPLES Example I Construction of Bacterial Strains

New E. coli host strains comprising at least one poly(A) polymerase geneoperably joined to a conditionally inducible promoter were constructedusing E. coli strain TransforMax™ EC100™-T1®(F-mcrA Δ[mrr-hsdRMS-mcrBC]80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsLnupG), which is commercially available from EPICENTRE.

E. coli strain TransforMax™ EC100™-T1®, which contains a wild-type pcnBgene, was mutated by a targeted knockout of said gene which ceases itswild type function by a homologous recombination technique facilitatedby the use of plasmid pKD46 which contains an IPTG inducible promoteroperably linked to genes encoding phage λ red, exo and bet proteins;said plasmid is available from the E. coli Genetic Stock Center (CGSC)at Yale University, CGSC No. 7739. The DHFR gene from the EZ::TN™<DHRF-1>Transposon (available from EPICENTRE) was amplified by PCR usingthe FailSafe™ PCR System to have 45 bases homologous to the pcnB gene atthe 5′ and 3 ends of the DHFR gene, henceforth called the pcnB-DHFRcassette, by use of the primers with the following sequences:

Forward: [Sequence I.D. No. 1]5′-CTCCAGCAGTTTCCATGCGCGTTTACCCTGACGACGGGACATACGCGGATAGACGGCATGCACGATTTG-3′; and Reverse: [Sequence I.D. No. 2]5′TCCGTAACTGCCCGCCTGGTGGGTCGCCGTTTCCGTCTGGCTCATGGCAGGTCGACTCTAGAGGATCCCCG-3′.

This pcnB-DHFR cassette was then processed by PEG precipitation,phenol/chloroform extraction and ethanol precipitation, and re-suspendedin 10 mm Tris; 1 mm EDTA (T₁₀E₁). Of the re-suspended DNA 200 ng wereelectroporated by standard techniques into the TransforMax™EC100™-T1®cell line containing the pKD46 plasmid. Homologousrecombinants of the wild type pcnB gene replaced with the pcnB-DHFRcassette were selected for on LB pates or Mueller-Hinton platessupplemented with 10 ug/ul trimethoprim. Recombinant clones werescreened by the phenotype of maintaining a pUC19 vector, a pBAD vector,a pET vector, a pCC vector, and a pHC vector at a low copy number.Recombinants were also analyzed by PCR using the FailSafe™ PCR systemwith primers specific for the DHFR gene, and by the ability to grow ontrimethoprim after sequential generations to ensure the stability ofpcnB-DHFR cassette. Once an TransforMax™ EC100™-T1®pcnB mutant clonethat had a reduced plasmid copy number, was able to grow on trimethoprimafter sequential generations, and contained a DHFR gene was selected,the pKD46 plasmid was removed by growing the mutant at the pKD46non-permissive temperature of 37° C. for sequential generations. Theremoval of the pdK46 plasmid was determined by the clones' inability togrow on ampicillin.

Then, the wild type pcnB gene was PCR amplified using the FailSafe™ PCRsystem from E. coli wild type strain MG1655 (available from the AmericanType Culture Collection (ATTC), ATTC No. 47076) genomic DNA usingprimers with the following sequences:

[Sequence I.D. No. 3] Forward: 5′-GCTATGATTAGCCGGAATTCTTTTGTCCTG-3′; and[Sequence I.D. No. 4] Reverse: 5′-CTGCCTATGGCAAGCTTCGCCACTGTCATG-3′.

This pcnB PCR product, henceforth referred to as the pcnB cassette, wasdigested with EcoRI and HindIII restriction enzymes and ligated into anEcoRI and HindIII digested sequence containing the araB promoter on aDNA fragment that contained an arabinose inducible operon, all withstandard molecular techniques, and clones selected for on ampicillin.Clones were screened initially by size; clones with the correct sizeinsert were grown up overnight, plasmid DNAs were mini-prepped andrestriction mapped to verify the correct insert. Once a clone wasselected to have the proper pcnB insert in the araB promoter containingDNA fragment, henceforth referred to as araB/pcnB, its mini-prep DNA wasthen digested with HindIII restriction enzyme and made blunt-ended usingthe End-It™ DNA End Repair Kit (EPICENTRE) according to themanufacturers protocol, and treated with shrimp alkaline phosphate toremove its free phosphate groups to prevent excess self-ligation.

Then a blunt ended DNA molecule with phosphorylated 5′ ends comprisingthe kanamycin resistance gene from the EZ::TN™ <KAN-2>Transposon whichis commercially available from EPICENTRE flanked by FLP recombinationtarget (frt) sites on its 5′ and 3′ ends, henceforth referred to as thefrt-kan cassette, was ligated into the HindIII, blunt, shrimp alkalinephosphate treated araB/pcnB construct and clones were selected for bythe ability to grow on LB agar supplemented with ampicillin+kanamycin.Clones were then grown overnight in LB broth supplemented withampicillin+kanamycin and mini-prepped to obtain DNA from each clone. Intwo different experiments clones were then restriction digested with twodifferent restriction endonucleases, BamHI and CIaI, to map theorientation of the frt-kan-frt cassette relative to the araB/pcnBconstruct. Once a clone was found in the 5′-3′ orientation relative tothe pcnB gene, the whole arabinose inducible operon comprising the araC,araB promoter, pcnB gene, frt-kan cassette, and the rrnBT₁T₂transcriptional terminator, henceforth referred to as the araBpcnBcassette, was PCR amplified using the FailSafe™ PCR system with primershaving the following sequences:

[Sequence I.D. No. 5] Forward: 5′-CGTCAATTGTCTGATTCGTTACC-3′ and[Sequence I.D. No. 6] Reverse: 5′-GAAGCATTTATCAGGGTTATTGTC-3′.

This araBpcnB cassette PCR product was blunted and kinased then ligatedinto an EcoRI, blunt ended, shrimp alkaline phosphate treated pMOD-2™plasmid (EPICENTRE) and selected for growth on LB agar supplemented withampicillin+kanamycin. Clones were then grown overnight in LB brothsupplemented with ampicillin+kanamycin and mini-prepped to obtain DNAfrom each clone. Clones were then restriction digested to map theorientation of the araBpcnB cassette relative to the pMOD-2™plasmid andthe correct clone with the araBpcnB cassette in 3′-5′ orientation withrespect to the pMOD-2™ plasmid was selected. An artificial Tn-5 basedtransposon containing the araBpcnB cassette was made by PCR using theFailSafe™ PCR system and multiple cloning site (MCS)PCR primers, whichare commercially available form EPICENTRE, comprising the followingsequences:

[Sequence I.D. No. 7] Forward: 5′-ATTCAGGCTGCGCAACTGT-3′ and[Sequence I.D. No. 8] Reverse: 5′-GTCAGTGAGCGAGGAAGCGGAAG-3′.

The artificial Tn5 based transposon was then prepared by PvuII digestionusing a protocol similar to one of the methods described for preparingEZ::TN™ Transposons in Product Literature No. 145 for the EZ::TN™pMOD-2™<MCS> Transposon Construction Vector (EPICENTRE). The resultingtransposon is used to prepare Transposome™ complexes as described in thesame Product Literature No. 145. Thousands of random insertion cloneswere obtained following electroporation of the transposome into themutant TransforMax™ EC100™-T1®pcnB⁻cell line, the creation was describedearlier in this example, and transposition clones were selected for onLB supplemented with trimethoprim+kanamycin. Random clones were thenchosen and analyzed for their ability induce to higher copy number uponaddition of arabinose, which then allows for transcription of the pcnBgene from the araB promoter.

Cultures were started with the TransforMax™ EC100™-T1R cell line as acontrol and with the pcnB-<BADpcnB> clone#4 cell line, a cell linecontaining the araBpcnB cassette. Each cell line contained one type ofplasmid from the following group of plasmids: pUC19, pET11a/rnhA (aPET11 plasmid with a polypeptide encoding DNA insert named “rnhA”),TOPO® TA vector, pMOD-2™<pCC-BAC> (a pMOD-2™ vector containing thepCC1BAC™ vector as a DNA insert) and a pHC79 cosmid. After overnightgrowth 2 ml of the each culture was diluted to 50 ml with fresh LB andgrown for 30 minutes. A sample from the cultures was taken for anuninduced sample. Then 200 μl of 10% arabinose was added to the rest ofthe cultures and the cultures were induced for 4 hours shaking at 37° C.

Plasmids were visualized from all cultures (control, uninduced, induced)for equal numbers of cells using a standard whole cell lysis protocol.Aliquots were run on agarose gels.

As shown in FIG. 1 the results indicated that the copy number of severalplasmids including pUC19, pET11a, TOPO® TA, pMOD-2™ and pHC79 wereincreased through arabinose induction.

1. An E. coli host cell for production of a foreign product, wherein theforeign product is selected from the group consisting of DNA, RNA and apolypeptide, wherein the foreign product is toxic, unstable, and/orinhibits propagation of the host cell when DNA that encodes said foreignproduct is present in the host cell in an R1-type or colE1-type plasmidat high copy number, wherein said host cell comprises a poly(A)polymerase gene that (a) is operably joined to a conditionally induciblepromoter, (b) is located in the chromosome of said host cell, and (c)catalyzes polyadenylation of a copA or RNAI antisense RNA molecule;wherein the host cell is obtained by (i) removing or inactivating aconstitutively-expressed poly(A) polymerase gene and introducing saidpoly(A) polymerase gene that is operably joined to theconditionally-inducible promoter into the chromosome of the host cell or(ii) operably joining the conditionally-inducible promoter to a poly(A)polymerase gene that is located in the chromosome of the host cell.
 2. Amethod for cloning and stably maintaining a foreign product in an E.coli host cell, wherein the foreign product is selected from the groupconsisting of DNA, RNA and a polypeptide, wherein the foreign product istoxic, unstable, and/or inhibits propagation of the host cell when DNAthat encodes the foreign product is present in the host cell at highcopy number, the method comprising, A. providing: (1) an E. coli hostcell for conditional control of copy number of an R1-type or colE1-typeplasmid, wherein said host cell comprises a poly(A) polymerase gene that(a) is operably joined to a conditionally inducible promoter, (b) islocated in the chromosome of said host cell, and (c) catalyzespolyadenylation of a copA or RNAI antisense RNA molecule; (2) an R1-typeor colE1-type plasmid; and (3) a DNA sequence encoding a foreignproduct; B. ligating the DNA sequence into the R1-type or colE1-typeplasmid to generate a recombinant plasmid; C. introducing therecombinant plasmid into the host cell; and D. growing the host cellcontaining the recombinant plasmid under conditions wherein saidconditionally inducible promoter that is operably joined to said poly(A)polymerase gene in said host cell is not induced, thereby cloning andstably maintaining the DNA sequence encoding the foreign product.
 3. Amethod for cloning, stably maintaining and generating a foreign productin an E. coli host cell, wherein the foreign product is selected fromthe group consisting of DNA, RNA and a polypeptide, wherein the foreignproduct is toxic, unstable, and/or inhibits propagation of the host cellwhen DNA that encodes the foreign product is present in the host cell athigh copy number, the method comprising: A. providing: (1) an E. colihost cell for conditional control of copy number of an R1-type orcolE1-type plasmid, wherein said host cell comprises a poly(A)polymerase gene that (a) is operably joined to a conditionally induciblepromoter, (b) is located in the chromosome of said host cell, and (c)catalyzes polyadenylation of a copA or RNAI antisense RNA molecule; (2)an R1-type or colE1-type plasmid; and (3) a DNA sequence encoding aforeign product; B. ligating the DNA sequence into the R1-type orcolE1-type plasmid to generate a recombinant plasmid; C. introducing therecombinant plasmid into the host cell; D. growing the host cellcontaining the recombinant plasmid under conditions wherein saidconditionally inducible promoter that is operably joined to said poly(A)polymerase gene in said host cell is not induced, thereby cloning andstably maintaining the DNA sequence encoding the foreign product; and E.contacting the host cell containing the recombinant plasmid with aninducing agent that induces expression of the poly(A) polymerase gene towhich the conditionally inducible promoter is joined, thereby increasingthe copy number of the recombinant plasmid and generating the foreignproduct.